Since the introduction of insulin in the 1920's, continuous strides have been made to improve the treatment of diabetes mellitus. Major advances have been made in insulin. purity, and availability. Various formulations with different time-actions have also been developed. Despite these improvements, subcutaneous injection therapy still falls short of providing the patient with convenient regulation and normalized glycemic control. Frequent excursions from normal glycemia levels over a patient's lifetime lead to hyper-or hypoglycemia, and long term complications including retinopathy, neuropathy, nephropathy, and micro- and macroangiopathy.
To help avoid extreme glycemic levels, diabetics often practice multiple injection therapy whereby insulin is administered with each meal. However, this therapy has not yet been optimized. The most rapid-acting insulin commercially available peaks too late after injection and lasts too long to optimally control glucose levels. Recently, considerable effort has been devoted to create insulin formulations and insulin analog formulations that alter the kinetics of the subcutaneous absorption process.
Because all commercial pharmaceutical formulations of insulin contain insulin in the self-associated state and predominately in the zinc-hexamer form, it is believed that the rate-limiting step for the absorption of insulin from the subcutaneous injection depot to the bloodstream is the dissociation of the self-aggregated insulin hexamer. Brange et al. in Diabetes Care 13: 923-954 (1990). To accelerate this absorption process, monomeric insulin analogs have been developed. These monomeric analogs possess a comparatively more rapid onset of activity than insulin while retaining the biological activity of native human insulin. They provide a rapid absorption to bring the injection time and peak action of insulin into closer proximity with postprandial glucose excursion associated in the response to a meal. The preparation of various monomeric analogs is disclosed in U.S. patent application Ser. No. 07/388,201 (Chance et al., EPO publication number 383 472), and Brange et al., EPO publication number 214 826.
unfortunately, the modifications to insulin, which cause these analogs to be monomeric, also result in a high rate of polymer formation in parenteral formulations. Because the expiration of insulin preparations occurs when levels of 1% polymer are obtained (U.S. Pharmacopoeia, 1990), minimizing this type of degradation is extremely important in reducing undesirable side effects. Therefore, it was desirable to formulate monomeric analogs in such a manner to cause the analog to self-associate to form a stable conformation, yet maintain its rapid absorption.
The addition of certain metal ions, primarily zinc, enhance the chemical stability by driving the insulin to associate and form hexamers, specifically the Zn(II)-T.sub.6 conformation. Further, phenolics have been shown to specifically bind to the insulin hexamer and induce an allosteric conformational change whereby the eight N-terminal amino acids of the B-chain are converted from the extended conformation to an alpha-helix. Derewenda, et al. Nature, 338: 594-596 (1989). This phenolic-bound conformation state is known as the Zn(II)-R state.
In stark contrast to these well-established observations that insulin readily aggregates in the presence of zinc to form well defined, stable zn-hexamer structure, early studies with monomeric insulin analogs revealed that any aggregation between zinc and the insulin analog is distinct from that observed with insulin. B. H. Frank, Text and Slide copies of Lecture given at the Conference on Insulin "Self-Association and Conformational Studies on Human Proinsulin and Insulin Analogs", University of York, (Aug. 29-Sep. 1, 1989). Further, the highly stable Zn-hexamer complex as seen with insulin is not observed with monomeric analogs. Id. Brems et al. Protein Engineering, 5:6, 527-533 (1992), disclose that monomeric Lys.sup.B28 Pro.sup.B29 -hI is less prone to dimerization and self-association to higher molecular weight forms than human insulin. Brems et al. continue to conclude that Asp.sup.B28 Pro.sup.B29 -hI, Ala.sup.B28 Pro.sup.B29 -hI, and Lys.sup.B28 Pro.sup.B29 -hI show little or no Zn-induced association and that Pro.sup.B29 insulin, Lys.sup.B28 insulin, Asp.sup.B28 insulin, and Ala.sup.B28 insulin demonstrate Zn-induced association, but less than Zn-insulin. Subsequent, unpublished experimental observations by the present inventors suggest that association with zinc is observed; however, such association between the analog and zinc differs from insulin. The association that is observed with these analogs is to a multitude of higher molecular weight forms and distinct from the predominate, well-defined, Zn-insulin hexamers. Therefore, it is clear that monomeric insulin analogs do not form the Zn(II)-T.sub.6 conformation in a manner analogous to insulin.
In view of the published literature, it is surprising that the present invention affords monomeric insulin analogs in a well defined, stable zinc-phenol hexamer complex. This hexamer complex is uniquely different from those complexes observed with insulin under identical conditions. Insulin complexes with zinc and phenol are in a Zn(II)-R.sub.6 conformation. The hexamer complex of the present invention is not identical to this conformation. Also quite remarkably, the insulin analog hexamer complex has a much greater propensity to dissociate than insulin. This propensity to dissociate translates into the desired fast-acting property.
Brange et al. in Current Opinion in Structural Biology 1:934-940 (1991) disclose various fast-acting stable insulin monomers and state that the obvious route to creating a fast-acting insulin is to prevent dimer or hexamer formation. Likewise, Brange et al. in Diabetes Care 13: 923-954 (1990) disclose that when insulin is administered as a hexamer, in addition to its slower free diffusion, the hexamer must be sterically more hindered than a monomer during the diffusion transport in the subcutis and/or during its passage through the capillary membrane. Further when injected subcutaneously, the Zn(II)-R.sub.6 conformation does not dissociate directly but must transform through the Zn(II)-T.sub.6 conformation. These conformational changes and the dissociation therefrom delay the onset of activity. Therefore, one skilled in the art at the time of invention believed that efforts to chemically stabilize the monomeric insulin analog with zinc by forming a well defined, hexamer complex would be unsuccessful, or if successful, would sacrifice the rapid onset of action desired.
The present formulation is a zinc-phenolic induced hexamer complex that is absorbed rapidly. The rate of absorption for the hexamer complex is at least two times that observed with insulin. Yet, when the hexamer complex is formulated, it is equally stable when compared to insulin against chemical degradation. Therefore, it is surprising that the present invention converts a monomeric insulin analog to a well-defined, stable zinc-phenol hexamer complex. Remarkably; when formulated, this hexamer complex retains the fast-acting properties associated with the monomeric insulin analog. Accordingly, the present invention provides a parenteral formulations of the insulin analog hexamer complex that is stable and fast-acting.